The vascular endothelium is critically important for human and mammalian physiology and pathology. The luminal plasma membrane of vascular endothelial cells, in particular, plays a major role in regulating permeability, coagulation/anti-coagulation processes, interactions with migrating cells in inflammatory processes, and metastasis of neoplastic cells. Furthermore, the luminal plasma membrane interacts with vasoactive substances and cytokines that both up-regulate cell adhesion molecules and control the plasminogen activator-inhibitor system. The membrane also interacts with plasma proteins, such as transferrin, albumin, and others, to internalize and selectively transcytose these substances.
In addition to the constitutive activities listed above, vascular endothelial cells have different morphologies and functions depending upon the type of tissue they serve. Some of these differences, which are reflected in their complement of cell surface proteins, appear to be established during embryogenesis and are maintained unless the tissue is altered during adult life. Alterations in surface proteins of endothelial cells from the same type of tissue also occur incident to tissue damage or pathology as in, for example, inflammation, ischemia, and neoplastic processes.
Solid tumors in particular are highly vascularized. Even though tumor-associated blood vessels are originally derived from normal blood vessels, their morphology differs significantly from that of normal tissue. The microvasculature of solid tumors is mainly composed of capillaries and some undifferentiated vessels lined by endothelial cells with relatively few adjacent pericytes or smooth muscle cells. By contrast, normal capillaries have ample pericytes, which are thought to reduce endothelial cell proliferation and induce capillary maturation. Tumor capillaries also differ from normal blood vessels in possessing less basement membrane, reduced vessel stability, and increased vessel permeability relative to normal capillaries. These gross differences in structure and function are reflected in plasma membrane proteins that are expressed in tumor-associated vascular endothelial cells and are absent from normal endothelia.
There is a need in the art for reagents that detect disease-specific components, e.g. components of tumor-associated vasculature, for use as diagnostic probes. Furthermore, the same components can be used as targets for therapeutic reagents that will destroy tumor-associated blood vessels and thereby starve the tumor of critical oxygen and nutrients. In addition, intravasation of cancer cells, which takes place on tumor blood vessels, can be blocked by the same means. Finally, vascular targets can be used to deliver various medicaments such as chemotoxins to the immediate environment of a tumor.
The possibility of targeting tumor-associated blood vessels in order to inhibit the growth of tumors has now been tested in a model system. Burrows et al., Canc. Res. 52:5954-5962, 1992. When neuroblastoma cells transfected with gamma interferon genes were injected subcutaneously into mice, the tumors that formed produced gamma interferon. The gamma interferon up-regulated cell surface MHC Class II antigens in the vascular endothelial cells within the tumor. When these mice were subsequently administered a hybrid immunotoxin that recognized MHC Class II antigens, the tumors in the animal model were markedly reduced in size, without apparent damage to other organs. The above-described results, however, do not indicate which endothelial cell surface proteins might be up-regulated during tumor development in a natural condition. Nor do they suggest a way for distinguishing endothelial surface antigens that are expressed in response to the tumor itself from endothelial surface antigens expressed in any rapidly proliferating endothelium.
A number of other researchers have expended considerable effort (using a variety of experimental techniques) in an attempt to identify tumor-specific endothelial antigens. Several groups have used monoclonal antibodies (MAbs) raised against different source cells to probe normal and tumor-associated endothelia. Murray et al., (Radiother. Oncol. 16:221-234, 1989) demonstrated that MAb MECA-20 stains tumor-associated endothelia, but also effectively stains the vasculature of normal tissue. Rettig et al. (Proc. Natl. Acad. Sci. USA 89:10832-10836, 1992), using MAb FB5, showed that the antigen, which they termed endosialin, is present in tumor-associated endothelium but absent from normal endothelium. Endosialin is, however, synthesized at high levels in cultured fibroblasts, which strongly suggests that it is a proliferation-associated protein, and therefore of limited use as a target of tumor-associated vasculature. Similarly, Schrappe et al. (Canc. Res. 51:4986-4993, 1991) reported that a chondroitin sulfate proteolglycan recognized by MAb 9.2.27 is present in cultures of tumor cells as well as in tumor-associated endothelia but this molecule is also present in proliferating endothelia. Finally, Wang, J. M. et al., Int'l J. Canc. 54:363-370, 1993) reported that MAb E-9 stained endothelia that were tumor-associated, embryonic, or regenerating, and therefore did not specifically bind to tumor-associated endothelia. It is currently believed that the E-9 antigen, and possibly other proteins detected by the MAb technique, are in fact endoglin, a previously isolated and cloned polypeptide expressed at various levels in all endothelial cells except those in bone marrow (Gougas, A. et al., J. Biol. Chem. 265:8361-8364, 1990).
Clarke and West (Electrophoresis 12:590, 1991) used radio-iodination to label the cell surface of cultured endothelial cells exposed to different proliferative and tumor-derived stimuli in an attempt to isolate tumor-associated proteins. The majority of the proteins induced by tumor-conditioned medium were also shown to be induced by proliferative stimuli, although in that paper, three tumor-specific protein species were said to have been detected. However, there has been no follow-up report of any particular truly tumor-specific antigens. Moreover, the radio-iodination technique has inherent limitations: it can only detect a relatively small subset of cell surface proteins (those having accessible tyrosine residues). If radioiodination cannot be detected in a test sample, this may mean that the protein is absent or that the protein is not labelled. This is a particular impediment when searching for tumor-specific endothelial surface proteins, because they are likely to be bound to growth factors and extracellular matrix components and thus masked from radioiodination. In addition, radioiodination probes can penetrate between endothelial cells to label nonendothelial cell proteins. Consequently, a protein that is iodinated is not necessarily made by endothelial cells. Furthermore, because only a small percentage of any one protein would be labeled by iodine, the labeling probe cannot be used in isolating the protein.
There is also a need in the art for reagents that detect other pathological changes in the surface of vascular endothelial cells, such as those that result from hypoxia and/or ischemia. Hypoxia is defined herein as deprivation of tissues of physiological levels of oxygen. Ischemia is the condition in which tissues are deprived of blood supply. In the case of heart attacks, an initial failure of blood flow leads to endothelial surface changes. This in turn initiates an inflammatory response in which neutrophils and other blood components adhere to the luminal surface of the endothelium, further constricting blood flow and exacerbating tissue damage. Identification of the relevant hypoxia-specific endothelial surface proteins would thus provide targets for diagnostic and therapeutic reagents. Agala, S. et al., Proc. Natl. Acad. Sci. (USA) 88:9897-9901, 1991 used two-dimensional gel electrophoresis to identify sixty apparently hypoxia-induced endothelial proteins, but their work did not discriminate between internal and surface proteins.
A method for physically isolating plasma membrane fractions from slime mold cells employing the colloidal silica technique (which the parent application has modified and applied to tissue) is disclosed by Chaney and Jacobson (J. Biol. Chem. 258:10062, 1983). In these experiments, however, the technique was not used to compare plasma membrane protein profiles of cells exposed to different proliferative or disease-mimicking stimuli, nor to identify disease-specific endothelial membrane molecules. Moreover, as will be explained further below, the art was resistant to use of endothelial cells in culture because of phenotypic drift and the possibility for artifact.